Temporal profiling of the adipocyte proteome during differentiation using a five-plex SILAC based strategy.
The adipose tissue has important secretory and endocrine functions in humans. The regulation of adipocyte differentiation has been actively pursued using transcriptomic methods over the last several years. Quantitative proteomics has emerged as a promising approach to obtain temporal profiles of biological processes such as differentiation. Stable isotope labeling with amino acids in cell culture (SILAC) is a simple and robust method for labeling proteins in vivo. Here, we describe the development and application of a five-plex SILAC experiment using four different heavy stable isotopic forms of arginine to study the nuclear proteome and the secretome during the course of adipocyte differentiation. Tandem mass spectrometry analysis using a quadrupole time-of-flight instrument resulted in identification of a total 882 proteins from these two proteomes. Of these proteins, 427 were identified on the basis of one or more arginine-containing peptides that allowed quantitation. In addition to previously reported molecules that are differentially expressed during the process of adipogenesis (e.g., adiponectin and lipoprotein lipase), we identified several proteins whose differential expression during adipocyte differentiation has not been documented previously. For example, THO complex 4, a context-dependent transcriptional activator in the T-cell receptor alpha enhancer complex, showed highest expression at middle stage of adipogenesis, while SNF2 alpha, a chromatin remodeling protein, was downregulated upon initiation of adipogenesis and remained so during subsequent time points. This study using a 5-plex SILAC to investigate dynamics illustrates the power of this approach to identify differentially expressed proteins in a temporal fashion.
Molina
Henrik
H
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.|Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.
Yang
Yi
Y
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.|Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.
Ruch
Travis
T
Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.
Kim
Jae-Woo
JW
Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.
Mortensen
Peter
P
Center for Experimental BioInformatics (CEBI), Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
Otto
Tamara
T
Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.
Nalli
Anuradha
A
Institute of Bioinformatics, International Technology Park, Bangalore 560066, India.
Tang
Qi-Qun
QQ
Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.
Lane
M Daniel
MD
Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.
Chaerkady
Raghothama
R
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.|Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.|Institute of Bioinformatics, International Technology Park, Bangalore 560066, India.
Pandey
Akhilesh
A
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.|Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.|Departments of Pathology and Oncology, Johns Hopkins University, Baltimore, MD 21205, USA.
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism*; D050156:Adipogenesis; D000818:Animals; D001391:Azo Compounds / Q000494:pharmacology; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D005786:Gene Expression Regulation; D007554:Isotopes; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D040901:Proteomics / Q000379:methods*; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D001391:Azo Compounds; D007554:Isotopes; D014357:Trypsin; C011049:oil red O